Abstract Zika virus (ZIKV) is a positive sense strand RNA virus within the Flaviviridae family of viruses that is currently on the brink of a global epidemic. ZIKV infection has been highly associated with the induction of neurological disorders, including Guillain-Barr syndrome and microcephaly. Yet, very little is known about the ZIKV-host interactions that regulate the outcome of infection. Recently, we demonstrated that infection of HIV is regulated by the dynamic, post-transcriptional RNA modification N6-methyladenosine (m6A). Importantly, m6A is a potent regulator of RNA function in humans and viruses. This project aims to determine how m6A affects the replication of ZIKV, both at the level of the viral RNA genome and at the level of the host mRNA. Our central hypothesis is that m6A acts directly on the ZIKV RNA genome and on a specific set of mRNAs to regulate ZIKV replication. The rationale for the proposed research is that by understanding how m6A regulates ZIKV replication, we can manipulate and target m6A-targeted processes to design new approaches for the prevention and treatment of ZIKV infection. Guided by our preliminary data, our hypothesis will be tested by pursuing these two specific aims: 1) Identify the sites of m6A within the ZIKV RNA genome at single nucleotide resolution, and 2) define the ZIKV infection-induced m6A profile on host mRNAs in the context of other Flaviviruses. In the first aim, we will define the life-cycle kinetics and sites of adenosine methylation on the ZIKV RNA genome at single nucleotide resolution by using m6A individual-nucleotide-resolution cross-linking and immunoprecipitation and direct RNA sequencing. For the second aim, we will perform a comparative analysis of virally-induced m6A changes in the host transcriptome following infection by Flaviviridae viruses, including ZIKV, hepatitis C, yellow fever, and dengue virus. This aim will define the conserved and unique host transcriptome and epitranscriptome responses to each viral infection. Taken together, these experiments will identify targets that define how m6A regulates ZIKV infection and disease, which represents a completely unknown molecular feature of this newly emerging virus. Ultimately, a detailed understanding of ZIKV biology, including how m6A affects ZIKV infection, will uncover novel strategies to develop antiviral therapies to target this RNA regulatory control that is exploited by RNA viruses for their replication.